P413: GENOMIC AND TRANSCRIPTOMIC ANALYSES OF NORMAL KARYOTYPE PRIMARY REFRACTORY ACUTE MYELOID LEUKAEMIA REVEAL HIGH INCIDENCE OF WT1 MUTATIONS AND A TRANSCRIPTIONAL SIGNATURE OF SENESCENCE.
Authors: Ali Alhawaj, Lucia Conde, Panagiotis D Kottaridis, Rosemary E Gale, David L Linch, Marc Mansour
Journal: HemaSphere (2023)

Background: In adults with acute myeloid leukaemia (AML), certain genetic abnormalities, such as a complex karyotype and/or mutations in TP53, are well-recognized to be associated with failure to achieve remission after induction chemotherapy, so-called induction failure (IF). Despite IF occurring up to 10% of normal karyotype (NK-AML), the genetic and transcriptional basis of chemotherapy resistance in this subgroup is less well characterized (1). Previous studies on small patient groups did not identify clear associations of IF with genetic mutations in NK-AML, emphasizing the need for a more comprehensive genomic analysis utilizing a larger sample size (2). Aims: To identify genomic driver mutations and transcriptomic signatures in NK-AML IF through analysis of primary patient samples. Methods: DNA samples from 70 NK-AML patients who experienced IF, and 70 matched NK-AML patients who achieved remission, were sequenced using the All Exon V6+UTR capture probes (Agilent) using the NovaSeq platform to reach a mean depth of 100X. Variant calling was done by the GATK 4.1.9 bundle. Paired-end mRNA sequencing was performed at 30 million reads using Illumina Novaseq platform on 43 NK-AML IF samples vs 48 chemoresponsive samples (n=48) matched for karyotype, age, sex, sample source, NPM1 and FLT3 mutational status. RNAseq analysis was performed by Nextflow nf-core RNAseq pipeline. Results: In IF NK-AML, the top mutated genes from WES were DNMT3A (39%), FLT3-ITD (33%) and NPM1 (29%; Fig 1). Most strikingly, WT1 mutations were present in 29% of IF patients and had a high mutual co-exclusivity with DNMT3A mutations (p<0.05), consistent with their opposing roles in DNA methylation. TP53 mutations were surprisingly rare, present in only 2 patients with IF. Unexpectedly, APC mutations occurred in 15.7% of IF patients. Gene-set enrichment analysis yielded a significant senescence signature in IF patients from two different datasets in The Broads Institute’s Molecular Signatures Database v7.5.1 (NES>2, FDR<0.01). This was supported by the enrichment of the accompanying senescence-associated secretory phenotype (SASP; NES=2.7, FDR<0.01) and quiescence signature in the IF group (NES=2.2, FDR<0.01). While previous studies have implicated chemotherapy-induced senescence as a mechanism of chemotherapy resistance (3), our study suggests a senescence phenotype can occur prior to chemotherapy leading to IF in a subset of patients. We speculate this is genetically associated with the high incidence of WT1 and APC mutations in this group of patients, given both genes have been implicated in regulating cellular senescence in other disease models (4-5). Furthermore, this study identifies a genetic and transcriptional signature of patients with NK-AML likely to experience IF that might prove a useful biomarker if confirmed on independent cohorts. Summary/Conclusion: • WES analysis in NK-AML IF identified a high incidence of WT1 mutations that are mutually exclusive with DNMT3A mutations. • APC mutations were unexpectedly enriched in NK-AML IF patients. • RNAseq revealed the enrichment of senescence and quiescence in NK-AML IF that proceeds chemotherapy. References: 1. Gaidzik V. Semin Oncol. 2008;35(4):346-55. 2. Brown FC. Br J Haematol. 2017;176(1):86-91. 3. Duy C. Cancer Discov. 2021;11(6):1542-61. 4. Hernandez-Segura A. Trends Cell Biol. 2018;28(6):436-53. 5. Vicent S. J Clin Invest. 2010;120(11):3940-52.Keywords: Senescence, Genomics, Chemoresistance, AML